The present invention relates to a novel tricyclic compound useful as a pharmaceutical agent to pharmaceutical compositions which include this compound and a pharmaceutically acceptable carrier, and to pharmaceutical methods of treatment. The novel compound of the present invention is an inhibitor of matrix metalloproteinases, e.g., gelatinase A (72 kDa gelatinase). More particularly, the novel compound of the present invention is useful in the treatment of multiple sclerosis and other autoimmune or inflammatory disorders dependent on the tissue invasion of leukocytes or other activated migrating cells.
Gelatinase A and stromelysin-1 are members of the matrix metalloproteinase (MMP) family (Woessner J.F., FASEB J. 1991;5:2145-2154). Other members include fibroblast collagenase, neutrophil collagenase, gelatinase B (92 kDa gelatinase), stromelysin-2, stromelysin-3, matrilysin, collagenase 3 (Freije J.M., Diez-Itza I., Balbin M., Sanchez L.M., Blasco R., Tolivia J., and Lopez-Otin C. J. Biol. Chem., 1994;269:16766-16773), and the newly discovered membrane-associated matrix metalloproteinases (Sato H., Takino T., Okada Y., Cao J., Shinagawa A., Yamamoto E., and Seiki M., Nature, 1994;370:61-65).
Matrix metalloproteinases share high sequence homology and the catalytic domains of each of the MMPs can be identified by sequence alignment. The gene for the catalytic domain of stromelysin-1, SCD, was constructed by removing the propeptide and C-terminal domain (Ye Q.-Z., Johnson L.L, Hupe D.J., and Baragi V., "Purification and Characterization of the Human Stromelysin Catalytic Domain Expressed in Escherichia coli", Biochemistry, 1992;31:11231-11235). The gelatinase A catalytic domain, GCD, was similarly constructed with the additional removal of the fibronectin-like insert which interrupts the catalytic domain (Ye Q.-Z., Johnson L.L, Yu A.E., and Hupe D., "Reconstructed 19 kDa Catalytic Domain of Gelatinase A is an Active Proteinase", Biochemistry, 1995;34:4702-4708). Both truncated proteins cleave synthetic peptide substrates and the natural substrates proteoglycan and gelatin in a manner similar to the full-length enzymes and can be used to identify matrix metalloproteinase inhibitors.
The catalytic zinc in matrix metalloproteinases is the focal point for inhibitor design. The modification of substrates by introducing chelating groups has generated potent inhibitors such as peptide hydroxymates, thio-containing peptides, and N-carboxyalkyl peptides. Peptide hydroxymates and the natural endogenous inhibitors of MMPs (TIMPs) have been used successfully to treat animal models of cancer and inflammation. However, except for amino acid derivatives with weak potency (Ye Q.-Z., Johnson L.L., Nordan I., Hupe D., and Hupe L., J. Med. Chem. 1994;37(1):206-209), few non-peptide inhibitors have been described or shown to have in vivo activity.
The ability of the matrix metalloproteinases to degrade various components of connective tissue makes them potential targets for controlling pathological processes. Gijbels, et al., (J. Clin, Invest. 1994;94:2177-2182) recently described a peptide hydroxymate, GM6001, that suppressed the development or reversed the clinical expression of experimental allergic encephalomyelitis (EAE) in a dose dependent manner, suggesting the use of MMP inhibitors in the treatment of autoimmune inflammatory disorders such as multiple sclerosis.
Multiple sclerosis (MS) is a complex demyelinating disease of the central nervous system (CNS) characterized by inflammation, disruption of the bloodbrain barrier, selective destruction of the myelin sheaths with glial scar formation and loss of neuronal cell conductivity leading to neurological deficits. The underlying cause is unknown, but it has been established as a T-cell mediated autoimmune disease (Lawrence Steinman, "Autoimmune Disease", Scientific American, Sep. 1993;269(3):106-114). While there are no spontaneous animal models for the disease, experimental allergic encephalomyelitis (EAE) has been used successfully to study many aspects of MS pathogenesis, and the work of Paterson and others has clearly and convincingly demonstrated the validity of this model as the only accepted preclinical test for efficacy of agents in MS (Paterson P., "Going to the Rats and Dogs to Study the Patient," Cell-Immunol., 1983;82(1):55-74). Bornstein's work using mammalian organotypic cultures showed that the CNS tissue responded with identical patterns of demyelination, swollen myelin sheaths, and eventual "sclerosis" when exposed to serum from EAE-affected animals and MS patients (Bornstein M.B., Miller A.I., Slagle S., Arnon R., Sela M., and Teitelbaum D., "Clinical Trials of Copolymer I in Multiple Sclerosis," in Annals of the New York Academy of Sciences, eds Labe Scheinberg and Cedric S. Raine, 1984;36:366-372). Analysis of the receptors on T-cells isolated from brain lesions of MS patients reveal that they are reactive to a peptide fragment of myelin basic protein analogous to the antigen used to precipitate the EAE model (Lawrence Steinman and Paul Conlon, "Designing rational therapies for multiple sclerosis," Bio/technology, Feb. 1995:118-120).
Several successful therapeutic strategies for treating MS target the T-cell response modeled in EAE. Beta-interferon (betaseron), acts in part by downregulating the expression of histocompatibility locus antigen (HLA) DR2. EAE studies have been used as the primary experimental basis to develop many of the current treatments for MS. Copolymer-1 and oral administration of myelin basic protein act by inducing immune tolerance to the myelin basic protein (MBP) antigen. The EAE model was used to develop a therapy in which peptides derived from the T-cell receptor V region recognizing an MBP fragment are used to immunize patients with relapsing-remitting MS. Monoclonal antibodies (Mab) against the CD4 receptor prevented the clinical and histological manifestations of EAE (Steinman L., Lindsey J.W., Alters S., and Hodgkinson S., "From treatment of experimental allergic encephalomyelitis to clinical trials in multiple sclerosis," Immunol-Ser., 1993;59:253-60). Clinical trials of CD4 Mabs are being conducted by several companies.
In MS and EAE, the blood-brain barrier has been shown to be defective both with respect to exclusion of blood-borne substances from the CNS and infiltration of lymphocytes (Lam D.K.C., "The central nervous system barrier in acute experimental allergic encephalomyelitis," in The Blood Brain Barrier in Health and Disease, edited by Suckling A.J., Rumsby M.G., and Bradbury M.W.B., 1986:158-164, Ellis Horwood, Ltd., Chichester, UK). Using gadolinium-DTPA enhanced magnetic resonance imaging (MRI), Miller, et al. (Miller D.H., Rudge P., Johnson G., Kendall B.E., MacManus D.G., Moseley I.F., Barnes D., and McDonald W.I., "Serial gadolinium enhanced magnetic resonance imaging in multiple sclerosis, " Brain, 988;111:927-939) showed in a serial study of MS patients that in recognizable new lesions or in new parts of existing lesions, blood-brain barrier impairment was always present. It appears that bloodbrain barrier disruption is the beginning of an irreversible cascade of events leading to demyelination (Barkhof F., Hommes O.R., Scheltens P., and Valk J., "Quantitative MRI changes in gadolinium-DPTA enhancement after high-dose intravenous methylprednisolone in multiple sclerosis," Neurology, 1991;14:1219-1222) and is necessary for the development of the disease (Moor A.C.E., De Vries H.E., De Boer A.G., and Breimer D.D., Biochemical Pharmacology, 1994;47:1717-1724). Previously, it has been shown that neutralizing antibodies to cell adhesion molecules prevent lymphocyte infiltration in the EAE, and that this inhibits the inflammatory response initiating the encephalomyelitis (Yednock T.A., Cannon C., Fritz L.C., Sanchez-Madrid F., Steinman L., and Karin N., "Prevention of experimental autoimmune encephalomyelitis by antibodies against .alpha.4 .beta.1 integrin," Nature, Mar. 5, 1992;356(6364):63-66). It has therefore been predicted that agents that prevent such infiltration may be among the most inviting prospects for therapy in MS.
The mechanism through which the blood-brain barrier is disrupted during the pathogenesis of MS and other inflammatory diseases of the central nervous system is under intense study. Rosenberg, et al. showed that activated gelatinase A injected intracerebrally attacks extracellular matrix and opens the blood-brain barrier. Treatment with TIMP-2 reduced the proteolysis and protected the blood-brain barrier. (Rosenberg G.A., Kornfeld M., Estrada E., Kelley R.O., Liotta L., and Stetler-Stevenson W.G., "TIMP-2 reduces proteolytic opening of the blood-brain barrier by type IV collagenase", Brain Research, 1992;576:203-207). A recent study by Madri has elucidated the role of gelatinase A in the extravasation of T-cells from the blood stream during inflammation (Ramanic A.M., and Madri J.A., "The Induction of 72-kD Gelatinase in T Cells upon Adhesion to Endothelial Cells is VCAM-1 Dependent", J. Cell Biology, 1994;125:1165-1178). This transmigration past the endothelial cell layer is coordinated with the induction of gelatinase A and is mediated by binding to the vascular cell adhesion molecule-1 (VCAM-1). Once the barrier is compromised, edema and inflammation are produced in the CNS. Leukocytic migration across the blood-brain barrier is known to be associated with the inflammatory response in EAE. Inhibition of the metalloproteinase gelatinase A would block the degradation of extracellular matrix by activated T-cells that is necessary for CNS penetration.
These studies provide the basis for the expectation that an effective, bioavailable inhibitor of gelatinase A would have value in the treatment of diseases involving disruption of extracellular matrix resulting in inflammation due to lymphocytic infiltration.
We have identified a tricyclic compound that is an inhibitor of matrix metalloproteinases, particularly gelatinase A, and is additionally active in an allergic encephalomyelitis model and thus useful as an agent for the treatment of multiple sclerosis or other autoimmune or inflammatory diseases dependent upon tissue invasion by leukocytes.